structural diversity and differential transcription of the - genetics

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.105.051219

Structural Diversity and Differential Transcription of the Patatin MulticopyGene Family During Potato Tuber Development

Robert M. Stupar,* Karen A. Beaubien,* Weiwei Jin,* Junqi Song,* Mi-Kyung Lee,†

Chengcang Wu,† Hong-Bin Zhang,† Bin Han‡ and Jiming Jiang*,1

*Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706, †Department of Soil and Crop Sciences, Texas A&MUniversity, College Station, Texas 77843 and ‡National Center for Gene Research, Chinese Academy of Sciences, Shanghai 200233,

People’s Republic of China

Manuscript received September 16, 2005Accepted for publication November 6, 2005

ABSTRACT

The patatin multicopy gene family encodes the major storage protein in potato tubers and is organized asa single cluster in the potato genome. We sequenced a 154-kb bacterial artificial chromosome (BAC) clonecontaining a portion of the patatin gene cluster. Two putatively functional patatin genes were found in thisBAC. These two genes are embedded within arrays of patatin pseudogenes. Using a chromatin immuno-precipitation method we demonstrate that the dramatic increase of patatin gene expression during thetransition from stolons to tubers coincides with an increase of histone H4 lysine acetylation. We used 39rapid amplification of cDNA ends to profile expression of different patatin genes during tuber develop-ment. The profiling results revealed differential expression patterns of specific patatin gene groupsthroughout six different stages of tuber development. One group of patatin gene transcripts, designatedpatatin gene group A, was found to be the most abundant group during all stages of tuber development.Other patatin gene groups, with a 48-bp insertion in the 39-untranslated region, are not expressed in stolonsbut display a gradual increase in expression level following the onset of tuberization. These results demon-strate that the patatin genes exhibit alterations in chromatin state and differential transcriptional regula-tion during the developmental transition from stolons into tubers, in which there is an increased demandfor protein storage.

THE patatin genes in potato encode proteins thatcompose up to 40% of the soluble protein in the

tubers (Prat et al.1990). Thus, the patatin protein familyrepresents the primary storage protein in potato tubersand is a critical nutritional component. Patatin proteinsalso exhibit enzymatic function as lipid acyl hydrolasesand have been demonstrated to have both plant defenseand antioxidant activities (Strickland et al. 1995; Liuet al. 2003; Rydel et al. 2003; Shewry 2003; Sharma et al.2004).

It has been estimated that there are �10–18 copies ofthe patatin genes in each monoploid (12 chromo-somes) potato genome (Twelland Ooms 1988). Geneticmapping of the patatin genes indicates that all genesmap to a single locus on chromosome 8 (Ganal et al.1991). The patatin coding regions are highly homo-logous with one another (in most cases, .90% nucle-

otide sequence identity) and most patatin proteins areimmunologically and biochemically indistinguishable(Park et al. 1983; Mignery et al. 1984; Pots et al. 1999).The similarities between patatin gene copies have madeit difficult to study several aspects of this multicopy genefamily using traditional molecular techniques, such asgel blotting and isozyme analysis. Therefore, many ques-tions remained about this gene family, especially thoserelated to genomic structure, organization, and tran-scriptional regulation of different patatin gene loci.

Patatin genes are highly activated following tuberinitiation (Hendriks et al. 1991; Bachem et al. 2000).Patatin genes are mainly expressed in tubers, with sig-nificantly lower amounts of transcripts observed in othertissues (Prat et al. 1990). However, patatin gene expres-sion in nontuber tissues can be induced by sucrose treat-ment (Rocha-Sosa et al. 1989; Wenzler et al. 1989a,b).Therefore, the expression of patatin genes appears to beassociated with the presence of sucrose, but sucrose isnot believed to directly regulate patatin gene expression(Grierson et al. 1994).

Several studies have focused on the molecular basisof patatin gene regulation. Pikaard et al. (1987) foundthat two classes of patatin genes, distinguished bythe presence or absence of 22 nucleotides in the 59-untranslated region (UTR), are differentially expressed

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under the following accession nos.: BAC 14K07,accession no. DQ274179; the 39 RACE sequence data, accession nos.DQ274180–DQ274460; the 401 bp of the 39 coding regions for patatingene groups A–Q, accession nos. DQ274461–DQ274477; and the patatincomplete CDS sequence data, accession nos. DQ274478–DQ274502.

1Corresponding author: Department of Horticulture, University ofWisconsin, 1575 Linden Dr., Madison, WI 53706.E-mail: [email protected]

Genetics 172: 1263–1275 (February 2006)

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in roots and tubers. Class I transcripts (lacking the 22nucleotides) were found to be tuber specific while classII transcripts (bearing the 22 nucleotides) were foundin both tubers and roots, but at a lower abundance thanclass I forms. GUS reporter assays have been used tolocalize the expression patterns of the class I and IIpromoters. These assays confirmed the discovery byPikaard et al. and further revealed that different patatingenes may be expressed in different tissue and cell types(Prat et al. 1990).

Further studies of patatin gene promoters revealedthat a highly conserved 100-bp region containing tworepeat domains, the A-box and the B-box, is criticalfor the regulation of class I patatin gene expression(Grierson et al. 1994). The A-box and the B-box wereobserved to bind nuclear proteins that were speculatedto be transcription factors (Grierson et al. 1994). Theprotein believed to bind the B-box was recently identi-fied (Zourelidou et al. 2002). This DNA-binding pro-tein, named Storekeeper, is thought to be a regulator ofpatatin gene expression, possibly acting as a transcrip-tion factor (Zourelidou et al. 2002).

While much has been learned about the regulation ofpatatin genes as a family, no known studies have ad-dressed the question of the specific regulation of dif-ferent patatin gene copies. In this study, we attempted tobetter define the patatin multicopy gene family in termsof gene structure, organization, and transcriptionalregulation during tuber development. Using a combi-nation of cytological and molecular techniques, we havefound that patatin genes occupy a single major locus permonoploid genome that may consist of many pseudo-genes as well as functional genes. The transcriptionalactivation of patatin genes upon tuberization is accom-panied by a change in relative histone H4 lysine acetyla-tion. We have further found that the different copieswithin the patatin gene family exhibit differential expres-sion patterns dependent on both tissue type and tuberdevelopment stage.

MATERIALS AND METHODS

Plant materials: A haploid potato clone USW1 (2n ¼ 2x ¼24) derived from potato cultivar ‘‘Katahdin’’ was used forbacterial artificial chromosome (BAC) library constructionand fluorescence in situhybridization (FISH) mapping. Potatocultivar ‘‘Kennebec’’ (2n¼ 4x¼ 48) was used for chromatin im-munoprecipitation (ChIP) and all gene expression analyses.Kennebec plants were grown in a walk-in growth chamber atthe University of Wisconsin (Madison, WI) Biotron. The plantswere exposed to a 12:12-hr light:dark photoperiod underfluorescent and incandescent lights. Chamber temperaturewas 23.5� during light hours and 15.5� during dark hours.Relative humidity was held constant at 50%. Following shootemergence, plants were given watering treatments with 0.53strength Hoagland solution twice daily.

Sequencing and FISH mapping of BACs containing patatingenes: A BAC library was constructed from the BamHI-digested genomic DNA of haploid clone USW1, using pub-

lished protocols (Song et al. 2000). The BAC library wasscreened with radioactively labeled patatin cDNA clonepGM01 (Mignery et al. 1984) and patatin genomic clonePS20SB (Mignery et al. 1988). Positive clones were finger-printed as described (Tao et al. 2001). One BAC, 14K07, wassequenced as described (Feng et al. 2002). The BAC sequencewas annotated for genes using a combination of ab initio genefinders and sequence similarity evidence (Yuan et al. 2002).Gene finders used include Genemark.Hmm (Lukashin andBorodovsky 1998), Genscan, and Genscan1 (Burge andKarlin 1997). Genes with transcript evidence only wereannotated as encoding ‘‘expressed proteins.’’ Genes predictedsolely by ab initio gene finders were annotated as encoding‘‘hypothetical proteins.’’ Sequence analysis and homologysearches with known patatin genes were further performedusing MegAlign software from the Lasergene ’99 package(DNAStar, Madison, WI). FISH mapping and image process-ing were performed as described ( Jiang et al. 1995).

RNA isolation and gel blot analysis: Two biological repli-cates of 10 Kennebec plants each were grown sequentially inthe same growth chamber. Plants were monitored periodicallyfor signs of flower formation. Stolon tips and tubers wereharvested from the entire population 3–5 days following thefirst indications of flowering in the chamber. All tissues wereflash frozen in liquid nitrogen. Harvested tubers were mea-sured in length prior to flash freezing. Tissues were dividedinto six groups: stolon (no tuber formation; stolon tips wereharvested, including the apical tip and part of the subapicalregion), 1- to 5-mm tubers, 6- to 10-mm tubers, 11- to 15-mmtubers, 16- to 25-mm tubers, and 26- to 35-mm tubers. Largertubers were not identified at the times of harvest.

Total RNA was isolated from tissue of each of the six tuberdevelopmental stages by TRIzol extractions according to themanufacturer’s instructions (Invitrogen, Carlsbad, CA) withonly minor modifications. The aqueous TRIzol:chloroformextractions were precipitated with 0.5 vol isopropanol and0.5 vol 1.2 m sodium chloride, 0.8 m sodium citrate solution.Purified RNA samples were treated with 2.5 units of DNase Iand incubated at 37� for 15 min and then extracted withphenol:chloroform. Purified RNA samples were quantifiedand qualified using the Nanodrop spectrophotometer withversion 2.5.3 software (Nanodrop Technologies, Montchanin,DE) and agarose gel electrophoresis analyses, respectively.

Total RNA samples from the two biological replicates ofstolon and tuber tissues were used as targets for RNA gel blotanalysis. RNA samples were run on a 1.2% agarose formalde-hyde denaturing gel and transferred to Hybond1 nylon mem-brane (Amersham Biosciences, Piscataway, NJ). Patatin cDNApGM01 (Mignery et al. 1984) and potato ubiquitin ESTSTMGC93 (provided by C. R. Buell at the Institute forGenomic Research) were used as probes. All probe DNA was32P labeled using the Strip-EZ DNA labeling system, accordingto the manufacturer’s instructions (Ambion, Austin, TX). Thegel blot membrane was prewashed in 65� Church buffer (7%SDS, 0.5 m Na2HPO4, 1 mm EDTA, pH 7.2) for a minimum of1 hr. The radioactive probes were denatured and thenhybridized to the membrane overnight at 65�. The membranewas sequentially washed with 23 SSC/0.1% SDS and 0.23SSC/0.1% SDS and exposed to X-ray film and developed. Themembrane was stripped and reprobed according to the Strip-EZ manufacturer protocol (Ambion).

ChIP and real-time polymerase chain reaction: Potatocultivar Kennebec plants were grown in walk-in growth cham-bers, as described above. Stolon tip and early tuber tissues (1–30 mm in length) were collected and flash frozen in liquid ni-trogen. ChIP was performed essentially as described (Nagaki

et al. 2003), using an antibody specific to histone H4 lysineacetylation (lys 5, 8, 12, and 16) (Upstate Biotechnology, Lake

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Placid, NY). Mock experiments using preimmunized rabbitserum served as nonspecific binding controls for each ChIPexperiment. Real-time polymerase chain reaction (PCR) withspecific primers was performed to analyze the relative enrich-ment of ChIPed DNA associated with histone H4 lysineacetylation in different genomic regions. All primers relatedto the patatin gene were designed from the BAC 14K07 se-quence. Real-time PCR reactions and subsequent ampliconmelting curve analyses were performed using the DyNAmoSYBR Green qPCR kit on a DNA Engine Opticon 2 real-timePCR detection system (Bio-Rad Laboratories, Hercules, CA).The comparative Ct method (Livak and Schmittgen 2001)was adapted to determine the relative enrichment of eachprimer region. A non-patatin region within BAC 14K07 wasused as the reference primer set (‘‘PrimerReference’’ in theequations below), as it displayed a lower enrichment of the H4lysine acetylation vs. mock treatment DNA in both stolons andtubers. Relative enrichment of H4 lysine acetylation for eachprimer set (‘‘PrimerX’’ in the equations below) was calculatedas follows:

Relative Enrichment ðREÞ ¼ 2^ðDCtPrimerX �DCtPrimerReferenceÞ;

where

DCtPrimerX ¼CtðMock DNAPrimerXÞ�CtðAcetylated DNAPrimerXÞ

and

DCtPrimerReference ¼ CtðMock DNAPrimerReferenceÞ� CtðAcetylated DNAPrimerReferenceÞ:

The DCtPrimerReference values were derived from averagingtwo experimental replicates from the same ChIP DNA samplesfor stolons and tubers, respectively. The sameDCtPrimerReference

values for stolons and tubers were respectively applied in allprimer RE calculations. For all other primers,DCtPrimerX valueswere determined for two experimental replications from thesame ChIP DNA samples for stolons and tubers, respectively.These values were independently used to calculate RE. Stan-dard deviations for each primer set were determined from thetwo experimental replicates. Real-time PCR was repeated forany experimental replicate pair with a Ct difference .1.0.

Gene expression profiling using 39 rapid amplification ofcDNA ends: A universal patatin sense primer was developedfor 39 rapid amplification of cDNA ends (RACE) analysis (de-tails are provided in supplemental Figure 2 at http://www.genetics.org/supplemental/). This 27-bp sequence (TGTTGCTCTCATTAGGCACTGGCACTA) was identified on the sensestrand in the fifth exon, 401 bp before the stop codon inpatatin genes. Single-stranded 39 RACE-ready cDNAs weregenerated from the total RNA of each of the six developmentalstages of one bioreplicate of tuberization. The protocol forcDNA synthesis is as described by the BD SMART RACE cDNAamplification kit (BD Bioscience, Palo Alto, CA), except thatSuperscript III (Invitrogen) was used as the reverse transcrip-tase. All subsequent PCR reactions were performed with Promega(Madison, WI) Taq polymerase. PCR of the cDNAs using anintron-spanning patatin primer set (top primer, ACTGAAATGGATGATGCGTCTGAGGCTAA; bottom primer, CCAAAAGGTTACATAATCCAAGCACACAT; annealing temperature 60�)was performed to verify the quality of the cDNAs and confirmthe absence of DNA templates. RACE PCR reactions wereperformed according to manufacturer’s instructions (BDBioscience), using the 27-bp patatin universal sense primerand the SMART RACE cDNA amplification kit universal primerA mix. Nested PCR reactions were performed on these

amplicons, using the patatin universal sense primer and thenested universal primer A as specified by the manufacturer(BD Bioscience).

The 39 RACE amplicons were cloned using the pGEM-TEasy Vector System II cloning kit (Promega). Clones with in-serts of expected sizes (�600–700 bp) were then used astemplates in sequencing reactions. DNA sequencing tem-plates were derived from either the colony PCR amplicons orplasmid DNA preparations. Sequence reactions using BigDyeterminator chemistry (Applied Biosystems, Foster City, CA)were performed in both the forward and reverse orientationwith M13 universal primers. Sequenced nucleotides werecalled independently by two software packages, Editview 1.0(Applied Biosystems) and the SeqMan software from theLasergene ’99 package. Sequences and sequence assemblieswere manually edited when appropriate.

The amplicon sequences were assembled into patatin groupson the basis of identical sequence matches, using MegAlignsoftware from the Lasergene ’99 package. Relative groupmRNA abundance was estimated on the basis of the frequencyof each group sequence per developmental stage. Sequencealignments of the groups were performed, using default set-tings with CLUSTAL X v.1.81 software (Thompson et al. 1997).Phylogeny of the groups was analyzed by the neighbor-joiningmethod with CLUSTAL X v.1.81 (Saitou and Nei 1987;Thompson et al. 1997).

A PCR strategy was employed to generate sequence data forthe complete coding region of some abundant patatin tran-scripts. A PCR primer matching a frequently observed motifin the 39-UTR of several patatin genes was designed: ATTTACAACTACAACCCGAGACCTTGAAT. This primer and a near-universal class I patatin 59-UTR primer (CMAACAAAATTTAAAAACAMWTTGAACATTTGC, where M¼C and A and W¼A and T) were used to generate PCR amplicons of the completepatatin coding regions from the 1- to 5-mm and 26- to 35-mmtuber cDNAs. Amplicons were cloned into the pGEM-T plas-mid vectors as described above. Plasmids were purified andthen sequenced as described above.

RESULTS AND DISCUSSION

BAC clones containing patatin genes were mapped toa single location in the potato genome: We constructeda BAC library using a haploid clone (USW1) derivedfrom potato cultivar Katahdin. The library consists of43,008 clones. Pulsed-field gel electrophoresis of 20random clones indicated that the average insert size is�120 kb (Bresee 2002). On the basis of the clone num-ber and average insert size, we estimated that this BAClibrary provides �5.2 times coverage of the potato ge-nome (Bresee 2002). Sixteen positive clones were iden-tified initially when the BAC library was screened usingpatatin gene plasmids pGM01 and PS20SB as probes(Figure 1A). Restriction fingerprint analysis showedthat these clones fall into two contigs, consisting of 8and 3 clones, respectively. The remaining five BACs,which showed only a single band or few weak bands ingel blot hybridization, failed to align with either contig.

The potato genome is highly heterogeneous. It is notknown if the separation of the two contigs is caused bythe heterozygosity of the potato genomic sequences. Tobetter understand their relationship, one BAC from

Patatin Gene Family of Potato 1265D

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each contig was labeled for FISH analysis. BAC 34O11associated with contig 1 and BAC 9E06 associated withcontig 2 were labeled with different colors and simulta-neously hybridized to the metaphase chromosomesof potato haploid clone USW1. The two BACs weremapped to the same chromosomal location (Figure1B). This FISH result indicates that there is only onemajor patatin multicopy gene locus per monoploid(12-chromosome) potato genome. Contigs 1 and 2 mayrepresent separate patatin gene clusters within the samechromosome region. This is consistent with geneticmapping experiments that concluded that the potatopatatin locus is divided into discrete domains (Ganal

et al. 1991).Sequence analysis of a 154-kb BAC containing

patatin genes: To gain a better understanding of theorganization of the patatin genes we sequenced a BAC

clone, 14K07, selected from contig 1. BAC 14K07 wasselected for sequencing due to its large insert size(154 kb) and the high number of strong bands thathybridized to patatin gene probes in DNA gel blot anal-ysis (Figure 1A). Annotation of the sequence of BAC14K07 identified 11 genes related to patatin-codingregions (Figure 1C; Table 1) interspersed with severalregions related to transposable elements (see GenBankaccession DQ274179).

Both class I and class II patatin genes were found inBAC 14K07 (Table 1). Upstream regions with similarityto known patatin gene promoters are present in 5 of the11 patatin genes (Figure 1C; Table 1). Additionally, BAC14K07 contains four regions with similarity to knownpatatin gene promoters, but they are not located imme-diately upstream of any patatin genes. We termed theseregions ‘‘naked’’ patatin gene promoters. All 11 patatingenes have the same orientation (Figure 1C). However,only one of the naked patatin gene promoters is ori-ented in this direction; the other three naked promotershave the reverse orientation relative to the patatingenes. It is unknown if these reverse-orientated nakedpatatin gene promoters drive antisense patatin genetranscription.

Among the 11 patatin genes present in BAC 14K07,only 2 appear to be fully functional. One gene,StPat14K07.03 (Table 1; Figure 1C, region 3), has theappropriate patatin gene composition of seven exonsand six introns (Bevan et al. 1986; Mignery et al. 1988)and does not contain any in-frame stop codons or frame-shift mutations. This gene has a coding region that isnearly identical to several known patatin cDNAs. Thesecond gene, StPat14K07.15 (Table 1; Figure 1C, region15), is interrupted in the fifth intron by the BAC vectorcloning site (Figure 1C), but contains no in-frame stopcodons or frameshift mutations through the five exonspresent (Table 1). Both of these putative functionalgenes have large regions 59 upstream of the putativetranscription initiation sites that are similar to knownpatatin gene promoters. These promoter-similar re-gions include conserved CAAT boxes, core enhancers,and TATA boxes (Table 1).

The other patatin genes in BAC 14K07 appear to bestructurally altered and/or truncated or contain in-frame stop codons (Table 1). These genes are incapableof encoding patatin proteins; thus we refer to them aspatatin pseudogenes. The patatin multicopy gene familyhas long been known to contain genomic pseudogenes(Pikaard et al. 1986), which may have been functionalgenes prior to sequence degeneration. In general, se-quences 59 upstream to each of the patatin pseudogenesin BAC 14K07 do not appear to have intact promoters.Most 59 upstream sequences are either unrelated topatatin gene promoters or only partially similar toknown patatin gene promoters (Table 1), suggestingthat the pseudogenes are not likely transcribed. Thepseudogenes with the greatest similarity to the patatin

Figure 1.—Molecular and cytological characterization ofBAC clones containing patatin genes. (A) DNA gel blot ofHindIII restriction fragments from genomic DNA of potatohaploid clone USW1 (Lane 1) and 10 BACs probed with pa-tatin gene clone pGM01. Lane 2, 68O09; lane 3, 9E06; lane 4,16O21; lane 5, 23P06; lane 6, 46B04 (appeared as a singletonin fingerprint analysis); lane 7, 34O11; lane 8, 14K07; lane 9,47D05; lane 10, 55M11; lane 11, 20P13. (B) FISH analysis ofBACs 34O11 (red signal) and 9E06 (green signal) on potatohaploid USW1 (2n ¼ 2x ¼ 24). The two BACs cohybridize tothe same two loci, despite being assigned to different contigsin fingerprint analysis. (C) Sequence map of BAC 14K07. Se-quences similar to patatin gene exon reading frames are indi-cated by blue bars and sequences similar to known patatingene promoters are indicated by red bars. Horizontal arrowsindicate the directional 59/ 39 orientation of the reading framesand promoters, respectively. The numbers above the horizontalarrows correspond to the ‘‘Region’’ column in Table 1. Verti-cal arrows indicate the approximate positions of the primersused for ChIP analysis; the letters above the vertical arrowscorrespond to the ‘‘Position’’ column in Table 2.


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gene promoter are StPat14K07.08 and StPat14K07.10(Table 1). These two pseudogenes have 272 and 918 bpof promoter-similar sequence 59 upstream of the putativestart codons, respectively. Both of these promoter-similar regions maintain some features that may pro-mote pseudogene transcription, such as the CAAT box,core enhancer, and TATA box. However, these featuresare better conserved in the two putatively functionalgenes StPat14K07.03 and StPat14K07.15 (Table 1). Itis possible that the pseudogenes StPat14K07.08 andStPat14K07.10 were once functional patatin genes thathave recently become pseudogenes. Therefore, promoterstructural features are still present in these pseudogenes,which are not observed in the other patatin pseudogenesin BAC 14K07 (Table 1).

Sequence analysis of BAC 14K07 revealed a uniquestructure of the patatin gene family in which functionalpatatin genes are present within regions rich in patatinpseudogenes. This structure suggests that patatin genesmay evolve by a mechanism similar to the birth-and-death process described in plant pathogen resistancegene (R gene) cluster evolution (Michelmore andMeyers 1998). Patatin pseudogenes may act as a usefulsource of allelic variation that can become functionalthrough interallelic recombination and gene conver-sion events. Interestingly, patatin proteins have beenimplicated in plant defense responses (Stricklandet al. 1995; Sharma et al. 2004), which may explainpossible structure similarities between the patatin genefamily and R gene clusters. However, it remains unclearhow individual patatin genes or clusters are selected forover the course of evolution and what selective pressuresmay dictate this process.

The abundance of patatin gene transcripts increasesduring tuber growth: Potato variety Kennebec was usedfor gene expression profiling of patatin genes frompretuberizing stolons to midgrowth tubers (Figure 2A).Total RNAs from six developmental stages were col-lected and analyzed. RNA gel blot analysis was per-formed to determine the relative abundance of patatingene transcripts during the different developmentalstages of tuber growth. No visible signal was observedfrom the potato stolon RNA when a patatin cDNA clonepGM01 was used as a probe (Figure 2B). Reverse tran-scriptase PCR (RT–PCR) analyses indicated that thepatatin genes were expressed in stolons (data presentedbelow); however, the transcription level was insufficientto generate a strong visible signal on the RNA gel blot.RNA hybridization signals became visible at the onsetof tuberization and increased in intensity throughouttuber growth (Figure 2B), indicating that the transcrip-tion of the patatin genes gradually increases throughoutthe early stages of tuber growth. Similar results, in whichpatatin gene expression is first detectable at tuberinitiation and then subsequently increases throughouttuber growth, have previously been reported forin vitro tubers (Hendriks et al. 1991; Bachem et al.

2000). Here we have confirmed these results with tubersgrown in vivo.

Patatin genes exhibit enhanced histone H4 acetyla-tion during tuber development: It has been recognizedin numerous organisms that acetylation at the lysineresidues of histone H4 is specifically associated withtranscriptionally active chromatin (Strahl and Allis2000). To reveal the H4 acetylation dynamics of thepatatin genes during tuber development we conductedChIP analysis, using an antibody specific to histone H4lysine acetylation. Immunoprecipitated DNAs were iso-lated from both stolon and tuber tissues (see materials

and methods). A series of PCR primers were designedfrom different regions of BAC 14K07, including patatin-related and non-patatin regions (Table 2). The primerswere initially tested in PCR reactions on Kennebec ge-nomic DNA templates; all primer pairs produced asingle visible band of the expected size (supplementalFigure 1 at http://www.genetics.org/supplemental/).These primer pairs were then used in real-time PCRanalysis of the ChIP DNA to assess the relative H4 lysine

Figure 2.—Tuber developmental stages and patatin RNAgel blot analysis. (A) The six tuber developmental stages sam-pled in this study were based on tuber length (see materials

and methods). Examples of sample developmental stages areshown. Stage 1, Stolon; stage 2, 1–5 mm; stage 3, 6–10 mm;stage 4, 11–15 mm; stage 5, 16–25 mm; stage 6, 26–35 mm.The diameter of the coin is 24 mm. Mature tuber is shownon the right as a developmental reference in the growthchamber environment, but was not sampled in this study.(B) Patatin gene expression level increases throughout tuberdevelopment. The patatin transcripts at the different tuberdevelopmental stages were detected by RNA gel blot analysisusing a DNA probe from patatin cDNA pGM01. (C) Equalloading of total RNA was confirmed by gel staining for rRNAwith ethidium bromide (data not shown) and by reprobingwith a DNA probe from ubiquitin EST STMGC93.


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acetylation of the genomic regions in stolon and tubers,respectively.

We used a non-patatin gene primer pair ‘‘a’’ (Figure1C; Table 2) as a reference for all other primers becausethis primer represents a region that displayed a low rel-ative histone H4 lysine acetylation in both stolons andtubers and is thus effective as a negative control. TheChIP data showed that patatin gene-related regionsgenerally have more histone H4 lysine acetylation thanthe reference non-patatin gene region ‘‘a’’ (Figure 3).The histone H4 lysine acetylation level of patatin generegions relative to non-patatin regions appears to begreater in tubers than in stolons (Figure 3). These re-sults indicate that there are alterations in the histoneacetylation pattern of patatin gene regions during thedevelopmental transition from stolons to tubers. Fur-thermore, these changes in chromatin state coincidewith the upregulation of patatin gene expression.

The different patatin gene regions exhibited variablelevels of acetylation (Figure 3). Patatin exons 1 and 5 arehighly enriched in the ChIPed DNA. Interestingly, the39-UTR region displayed the greatest enrichment inboth the stolons and tubers. The only patatin gene re-gion that showed no obvious histone H4 lysine acetyla-tion enrichment was the third intron. It has been welldemonstrated in both plant and animal species thatthere may be significant variation of histone modifica-tion throughout different regions within a single gene(He et al. 2004; Schneider et al. 2004). However, in thepresent study the regional variation in relative enrich-ment within individual gene copies needs to be inter-

preted with caution because the number of loci actuallyrepresented by each primer pair is unknown. For in-stance, exons tend to be more conserved than intronsthroughout the patatin gene family; thus multiple exonloci may be contributing to the PCR products in boththe exon 1 and the 5 primer pairs; whereas the intron 3primer pair is more likely to be specific to one or a fewloci. Therefore, each primer pair may represent somerange of patatin gene loci, and the relative enrichmentobserved may reflect the chromatin alteration withinthat range of loci.

The 39-UTR structure of patatin genes is correlatedwith differential expression during tuber development:We used 39 RACE to profile the expression of patatingenes preceding and throughout tuber development(see materials and methods). A universal patatin genesense primer was developed for the 39 RACE analysis(supplemental Figure 2 at http://www.genetics.org/supplemental/). We sequenced a total of 278 patatin39 RACE amplicons, with a minimum of 43 sequencesfrom each of six developmental stages. The ampliconscan be broadly grouped into two categories on the basisof the presence or absence of a 48-bp region located inthe 39-UTR (Figure 4). The patatin genes reported inthe literature and found in BAC 14K07 display eitherform of this 39-UTR (Table 3). Therefore, the presence/absence of this 48 bp appears to be a consequence ofvariation in the DNA template rather than of differen-tial splicing in the 39-UTR. The stolon amplicons con-sisted exclusively of transcripts lacking the 48-bp region.A gradual increase in transcripts with the 48-bp region

TABLE 2

Primers for real-time PCR using ChIP DNA as templates

Positiona Gene specificityPrimer design

template Primer sequenceAmplicon

(bp)Annealing

temperature

a Non-patatin BAC 14K07 GCTAAAGGTGTTATTGAAACTGAGGAATC 242 60�TCTGAACTCTATGAAATGCTTGTTGAGAC

b Non-patatin BAC 14K07 GTCAGTCATTCCATTACTTGCCGAAACGA 255 60�CGTGTGACCACATTTTATGTAATAGGTAG

c Class I promoter BAC 14K07 gene 3 ATGGTAGATTGTTTGAGCGGATAATCTTC 252 60�GGTATATATAGGCACGTAAACAAGTTGAG

d Class II promoter BAC 14K07 gene 15 TGCTGCATTATTTCTTTTACATTGTTGGAT 304 60�ACTACAACAAGGAGTCGTTTGATAGAGTG

e Class I 59-UTR—exon 1

BAC 14K07 gene 3 CCAACAAAATTTAAAAACACTTTGAACATTTGC 200 60�AGTTGTCCTTCAAGAAATTCGAGAATGGT

f Class II 59-UTR—exon 1

BAC 14K07 gene 15 TTGAGATATCAGTTTTTATTAATTATAATCTGC 200 55�AGTTGTCCTTCAAGAAATTCGAGAATGGT

g Patatin intron 3 BAC 14K07 gene 3 GGTGAGATCGGACGTGTTTAGGAGTAGTT 231 65�AAATGAACCCATTCACCACTGAGATCCAC

h Patatin exon 5 BAC 14K07 gene 3 GATCCAGCATTTGCTTCAATTAGGTCATT 241 60�CCCTGAGGTAATTGTTTTGTGAATGAAGA

i Patatin 39-UTR BAC 14K07 gene 3 TTCAAGGTCTCGGGTTGTAGTTGTAAAT 147 55�TCAAACATTAAGCACAACTAAAAGGTTAC

NA Positive control Ubiq conj enzyme2 AGCTTTGATGACTGGAATCTTGTTTTGTG 269 65�CAACTCTGAGCCCTTCACAATAAAAGCAC

a Refers to the position indicated in Figure 1C.


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was observed from early through later tuber growth(Figure 5).

The amplicon sequences were further subdivided intogroups on the basis of sequence identity within the final401 bp of the 39 coding region. The 39-UTR sequenceswere not included as criteria in these groupings due tothe occasional occurrence of ambiguous sequence readsin these regions, in particular around the polyadeny-lated sequences. A total of 180 amplicons were groupedinto 17 different groups that are designated patatingene groups A–Q (Figure 6). Another 98 amplicons didnot have any identical sequence matches. The predictedamino acid sequences of the 17 groups were aligned andanalyzed phylogenetically (supplemental Figure 3A athttp://www.genetics.org/supplemental/). There was acorrelation between group sequence similarity andthe presence/absence of the 48 bp in the 39-UTR(supplemental Figure 3B at http://www.genetics.org/supplemental/). Therefore, the increased frequency oftranscripts with the 48 bp in the later tuber develop-mental stages suggests that there is an associated shift inthe relative abundance of certain patatin protein forms.

The cause and rationale for such a shift in differentdevelopmental stages remain unknown. Patatin genesshow tissue- and cell-specific transcriptional regulation.Pikaard et al. (1987) divided patatin genes into twoclasses on the basis of the presence (class II) or absence(class I) of 22 nucleotides in the 59-UTR and deter-

mined that the two classes are correlated with the tissuespecificity of patatin gene transcription. It was sub-sequently demonstrated that patatin gene promotersdetermine the cell-specific transcription of some patatingenes (Koster-Topfer et al. 1989; Rocha-Sosa et al.1989; Wenzler et al. 1989b). For example, a class I pro-moter showed transcriptional activity throughout tuberparenchyma cells (Rocha-Sosa et al. 1989), but expres-sion of a class II promoter was limited to fewer and morespecific cell types (Koster-Topfer et al. 1989). The rela-tive proportion of certain cell types will change through-out stolon and early tuber development (Artschwager

1924; Reeve et al. 1969). Therefore, the transcriptionalabundance of cell-type-specific patatin genes is proba-bly, in part, determined by the relative abundance of celltypes present at a given developmental stage. In thepresent study, this may explain the shift in later tuberdevelopment toward transcripts with the additional48 bp in the 39-UTR.

The presence/absence of the 48 bp in the 39-UTR andthe different 59-UTR classes characterized by Pikaardet al. (1987) appear to associate with gene expression indifferent ways. Previous studies showed that tubers ex-press class I patatin genes 50- to 100-fold more fre-quently than class II forms, implying that tubers maysilence certain patatin gene forms and selectively ex-press forms with specific 59-UTR structures (Pikaardet al. 1987; Mignery et al. 1988). However, in the present

Figure 3.—Histone H4 lysineacetylation patterns of patatingenes at two different develop-ment stages (stolons vs. tubers).The letters in parentheses repre-sent the PCR primer positions inBAC 14K07 (Figure 1C). All datawere standardized to the referenceprimer set for the primer pair (a).Error bars indicate the standarddeviation of two experimental rep-licates from the same ChIP DNAsamples. The horizontal dashedline represents a relative H4 lysineacetylation enrichment of 1, whichis the baseline enrichment of thenon-patatin (a) region. All values.1 indicate a relative fold increaseof histone H4 lysine acetylationcompared to the non-patatin (a)region (see Table 2 for real-timePCR primer information and sup-plemental Figure 1 at http://www.genetics.org/supplemental/ for PCRprimer pair quality assessment).


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study tubers expressed transcripts both with and withoutthe 48 bp in the 39-UTR, while stolons expressed onlythe forms lacking the 48-bp sequence. This suggests thattuberization is correlated with activation of patatin geneswith variable 39-UTR structures that appear to be si-lenced in stolons. Interestingly, on the basis of 59-UTRanalyses Pikaard et al. (1987) suggested that a specificsubset of patatin genes is predominant in tuber patatinexpression, but our data suggest that tuber patatinforms become more diverse following tuber initiationand growth (also see Table 4).

It is unlikely that the 59-UTR class structure is as-sociated with the presence/absence of the 48 bp in the39-UTR. As stated above, class I transcripts are �50- to100-fold more prevalent than class II transcripts intubers (Mignery et al. 1988). Therefore, we assume thatthe vast majority of our sampled 39 RACE amplicons areclass I forms, including those with the extra 48 bp inthe 39-UTR, which is approximately one-third of thetranscripts in the later tuber developmental stages(Figure 5). Additionally, structural analysis of genomicclones and full-length cDNAs indicated that the 59-UTRclass structure is not related to the presence/absence ofthe 48 bp in the 39-UTR (Table 3). Patatin gene struc-

tures have been identified for all four possible combi-nations: class I 59-UTR with the 48-bp 39-UTR sequence,class I 59-UTR without the 48-bp 39-UTR sequence, classII 59-UTR with the 48-bp 39-UTR sequence, and class II59-UTR without the 48-bp 39-UTR sequence (Table 3).

The 98 patatin 39RACE sequences that do not have anyidentical matches within the data set were designated as‘‘singletons.’’Fivesuchsingletonsequences(GenBank ac-cessions DQ274223, DQ274236, DQ274254, DQ274326,and DQ274337) have frameshift mutations within thecoding region, which may represent degenerated cod-ing regions of transcribed pseudogenes or may resultfrom experimental errors. Some singletons appear tobe unique patatin gene transcripts, with several uni-que base substitutions and no codon frameshifts (forexample, the sequence for clone ‘‘PCR30’’ in Figure 4appears to be a very unique functional singleton). How-ever, it is unlikely that all the singletons representunique patatin genes. Sequence identity comparisonswere made between the remaining 93 singleton se-quences and the 17 patatin gene groups from thisstudy. It was determined that 51 of the singletons(�55%) had only one nucleotide mismatch from beingidentical to one of the 17 groups. This indicates that

Figure 4.—Structural diversity of patatin genes in the 39-UTR. Shown here are eight examples of 39 RACE PCR amplicon se-quences amplified with the patatin universal sense primer. These amplicons were all sampled from the 1- to 5-mm developmentalstage. Polymorphic nucleotides are indicated with shading. The terminal nucleotide of the stop codon is marked with an arrow.The 48-bp 39-UTR insertion/deletion region is also marked by arrows. ‘‘PCR’’ notation indicates that the amplicon was derivedfrom a colony PCR template. ‘‘Plas’’ notation indicates that the amplicon was derived from prepped plasmid template. However,the template source should not affect the results (GenBank accession numbers for the displayed sequences: DQ274226,DQ274227, DQ274232, DQ274237, DQ274244, DQ274252, DQ274260, and DQ274266).


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many of the singletons may represent true groupmatches; however, they exhibit nucleotide polymorphismas a consequence of sequencing errors or PCR-inducedmutations.

The most abundant patatin gene group remainsdominant throughout tuber development: Despite theshift in transcript frequency correlated with the 39-UTRstructure, patatin gene group A (GenBank accessionDQ274461), which lacks the 48-bp 39-UTR insertion, isthe most abundant patatin gene group in all stages oftuber development (Figure 6). We found that 99 of the278 total amplicons are 100% identical to the patatingene group A sequence throughout the terminal 401-bpcoding region. However, it is unclear whether thesetranscripts are derived from a single locus or from mul-tiple loci with identical sequences in the final 401 bp of

their coding region. Patatin gene group A has no iden-tical sequence matches in GenBank. The most similarsequence match is a class I patatin cDNA from a Koreanpotato cultivar (GenBank accession Z27221). This se-quence is identical to 400 of the 401 coding bases inpatatin gene group A and has identical sequence matchesfor almost the entire 39-UTR. The next most similarpatatin gene match in GenBank is only�94% identical tothe coding region of group A.

We used PCR to amplify and sequence the entirecoding regions of the patatin gene group A transcript.We designed a primer matching the 39-UTR of severalpatatin gene groups, including group A (ATTTACAACTACAACCCGAGACCTTGAAT), and a near-universalclass I patatin 59-UTR primer (CMAACAAAATTTAAAAACAMWTTGAACATTTGC, where M ¼ C and A andW ¼ A and T) to amplify the entire coding regions of1- to 5-mm and 26- to 35-mm tuber cDNAs. A total of 34amplicons were cloned and sequenced. The entire codingregions were determined for these 34 clones; thus werefer to these data as patatin coding sequences (CDS).

Several different patatin CDSs were identified by thisapproach, including transcripts that match the terminal401 coding nucleotides from patatin gene groups A, D,M, and J (Table 5, GenBank accessions DQ274478–DQ274487). Additionally, 15 unique CDSs were identi-fied (GenBank accessions DQ274488–DQ274502); theseCDSs do not match any of the patatin gene groups iden-tified by 39 RACE. All 34 CDSs, however, display perfectconservation of the patatin gene universal sense 27-bppriming region used for generating the 39RACE libraries,further confirming the unbiased nature of the RACEprofiling approach used in this study.

Nine CDSs matched the terminal 401-bp codingregion of patatin gene group A (Table 5). Seven ofthese nine CDSs were identical throughout the entireCDS and were thus designated ‘‘patatin gene groupA-1’’ (GenBank accession DQ274478). Interestingly, the

Figure 5.—Relative abundance of patatin gene transcriptswith the additional 48 bp in the 39-UTR throughout tuber de-velopment. The proportion of 39-UTR types is based on rela-tive abundance per developmental stage.

TABLE 3

The 59- and 39-UTR structural features of sequenced patatin genes

Accession 59-UTR structure 39-UTR structure Source Reference

X01125 Class I 48 bp present cDNA Mignery et al. (1984)U09331 Class I 48 bp present cDNA Banfalvi et al. (1994)AF498099 Class I 48 bp present cDNA GenBank accessionX13179 Class I 48 bp present cDNA Stiekema et al. (1988)M21879 Class I 48 bp present cDNA Stiekema et al. (1988)X13178 Class I 48 bp present cDNA Stiekema et al. (1988)Z27221 Class I 48 bp absent cDNA GenBank accessionStPat14K07.03 Class I 48 bp absent Genomic gene This studyM18880 Class I 48 bp present Genomic gene Mignery et al. (1988)X03956 Class I 48 bp present Genomic gene Bevan et al. (1986)X03932 Class II 48 bp absent Genomic gene Rosahl et al. (1986)StPat14K07.08 Class II 48 bp present Genomic pseudogene This studyX04077 Class II 48 bp present Genomic pseudogene Pikaard et al. (1986)X04078 Class II �20 bp present Genomic pseudogene Pikaard et al. (1986)


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patatin gene group A-1 CDS is identical to the predictedcoding sequence of gene StPat14K07.03 from BAC14K07. The remaining two group A sequences eachhad one nonsynonymous nucleotide difference relativeto the patatin gene group A-1 sequence and were thusdesignated A-2 and A-3, respectively (GenBank acces-sions DQ274479 and DQ274480). It is unclear whetherthese polymorphic bases represent true nucleotidevariation or variation caused by technical error. Never-theless, it appears that the patatin gene group A-1 se-

quence (supplemental Figure 4 at http://www.genetics.org/supplemental/) at minimum represents the ma-jority of the group A transcripts. Thus, we propose thatthe patatin gene group A-1 represents the most abun-dant form of patatin found throughout tuber develop-ment in potato cultivar Kennebec.

It would be interesting to know why patatin genegroup A is so abundant throughout early tuber devel-opment. Patatin gene coding regions are highly con-served; however, patatin gene promoters are highlyvariable in terms of sequence motifs and structure. Dif-ferent patatin gene promoters show variable expressionabundance and localization in promoter-GUS fusionassays (Koster-Topfer et al. 1989; Rocha-Sosa et al.1989; Wenzler et al. 1989b). Therefore, it appears thatthe variations in patatin gene promoter sequence struc-ture ultimately determine the activity and function ofany given patatin gene. The patatin group A promotermay have features that confer the high activity of thisgene(s).

Comparison of patatin and maize zein gene families:The zein gene family of maize is one of the best-characterized storage protein gene families. ExtensiveBAC-based sequence analysis has been conducted on

Figure 6.—Relative abundance of differ-ent patatin gene groups throughout tuberdevelopment. Relative frequency of the17 patatin groups is displayed per develop-mental stage. Groups that have an addi-tional 48 bp in the 39-UTR (see Figures 5and 6) are denoted by asterisks (*); thesetranscripts become more abundant in thelater developmental stages. The 98 ‘‘single-ton’’ amplicons (described in results and

discussion) were not included in the rela-tive frequency calculation.

TABLE 4

Patatin gene groups detected in each developmental stage

Developmental stage Groups detected

Stolon (mm) A, B, C1–5 A, B, D, Ea, G, H, Oa

6–10 A, B, D, Ea, Fa, I, Pa, Q11–15 A, B, D, Ea, Fa, Ja, K, L16–25 A, D, Ea, Fa, Ja, Ma, N, Oa

26–35 A, D, Ea, Fa, Ma, N, Pa, Q

a Groups are associated with the additional 48 bp in the39-UTR.


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the zein gene clusters (Song et al. 2001; Song andMessing 2002, 2003). Sequence analysis of BAC 14K07indicates that the patatin gene clusters have similarstructural features to the zein gene clusters. Similar tothe patatin gene family, functional zein genes are inter-spersed with inactive zein genes (Song et al. 2001; Songand Messing 2002, 2003). These inactive forms havefeatures similar to the patatin pseudogenes describedabove, including gene truncation, internal deletion,and premature stop codons (Song et al. 2001; Song andMessing 2002). One extensively studied gene cluster,the 22-kDa a-zein cluster, is 346 kb in length andincludes six active genes and 16 copies inactive due toin-frame stop codons (Song et al. 2001). The zein genedensity in this cluster is �1 copy per 15.7 kb. The BAC14K07 sequence indicates that the patatin locus mayhave a similar patatin gene density and a similar ratio ofactive to inactive copies. Additionally, all active and in-active copies of the 22-kDa a-zein cluster are orientatedwith the same directional polarity (Song et al. 2001;Song and Messing 2003), which is also true of the BAC14K07 patatin gene copies.

The functional and structural similarities of patatinand zein gene families suggest that they may have ex-perienced similar evolutionary pathways. As discussedabove, patatin proteins likely have secondary functions,and evolutionary selection for these functions mayexplain the current patatin gene structure. The zein pro-tein clusters have similar structural features to patatin,but have not been shown to have a secondary functionbeyond seed protein storage. This suggests that storageprotein gene families may be under selective pressuredirectly for the protein storage trait. Differential gene

expression of zein gene copies in different genetic back-grounds (Song and Messing 2003) and of patatin genecopies throughout tuber development supports the hy-pothesis that each protein storage gene copy may dif-ferentially contribute to the plant phenotype and isthereby subject to selection for protein storage function.

Sequencing of 39 RACE amplicons as a tool fortranscript profiling of multicopy gene families: Theanalysis of transcriptional regulation of multicopy genefamilies is problematic in part because it is difficult todistinguish between gene copies with similar structureand sequence. Hybridization-based techniques, such asRNA gel blot or microarray analysis, cannot distinguishbetween the various transcripts from multiple loci.Quantitative real-time PCR is also of limited value forsimilar reasons; PCR primers may be difficult to designfor individual gene copies due to the high sequencesimilarity among the different gene members. Song andMessing (2003) were able to profile the expression ofthe zein gene family in maize by cloning and sequencingPCR amplicons from zein gene transcripts. Using cDNAtemplates, they performed PCR reactions with primersthat are conserved among all zein genes. Their PCRamplicons had sufficient internal polymorphism thatallowed different gene copies to be distinguished by se-quence analysis.

In this study, we were able to design one conservedpatatin sense primer toward the 39 end of the transcript.However, we were unable to design a compatible anti-sense primer because of sequence divergence amongknown patatin gene transcripts in the compatible re-gion. Therefore, the conserved sense primer was used togenerate 39 RACE amplicons, essentially exploiting thepolyadenylation tail as the antisense primer in the PCRreaction. The cloning and sequencing of these ampli-cons generated a data set from which transcripts couldbe analyzed and categorized on the basis of sequenceidentity and polymorphism. We were then able to utilizepriming sites from the 39-UTR sequence of abundanttranscripts and from a near-universal 59-UTR region toPCR amplify and sequence the complete CDS of themost transcribed patatin genes. Our results show thatthis strategy was effective in profiling the expression ofthe potato patatin gene family and can be potentiallyused to analyze other multicopy gene families.

We thank Bala Pudota for assisting in the growth of plant materials.We are grateful to Robin Buell, Jia Liu, and Bill Belknap for theircontributions in sequence annotation of BAC 14K07 and to RobinBuell for critical reading of this manuscript. This work was supportedby National Science Foundation Plant Genome Research Programgrants DBI-9975866 and DBI-0218166.

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TABLE 5

List of fully sequenced patatin coding sequences (CDS)

CDS name No. of clonesa Notes

Patatin A-1 7Patatin A-2 1 1 nt different from A-1Patatin A-3 1 1 nt different from A-1Patatin D-1 2Patatin D-2 1 1 nt different from D-1Patatin D-3 1 1 nt different from D-1Patatin M-1 3Patatin M-2 1 1 nt different from M-1Patatin M-3 1 1 nt different from M-1Patatin J-1 1Unique CDSsb 15

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b Unique CDSs have no identical matches to the patatingroups defined by the terminal 401 nucleotides or to anyother CDS in this study.


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